Pharmacological disruption of the Notch transcriptionfactor complexRajwinder Lehala,b, Jelena Zarica, Michele Vigoloa,b

, Charlotte Urechb, Viktoras Frismantasc,d, Nadine Zanggere,

Linlin Caoa, Adeline Bergerf, Irene Chicoteg, Sylvain Loubéryh, Sung Hee Choii, Ute Kocha

, Stephen C. Blacklowi,Hector G. Palmerg, Beat Bornhauserc,d, Marcos González-Gaitánh, Yvan Arsenijevicf, Vincent Zoetee,j, Jon C. Asterk,Jean-Pierre Bourquinc,d, and Freddy Radtkea,1

aSwiss Institute for Experimental Cancer Research, Ecole Polytechnique Fédérale de Lausanne, 1015 Lausanne, Switzerland; bResearch & Development,Cellestia Biotech SA, 4057 Basel, Switzerland; cDepartment of Oncology, University Children’s Hospital Zürich, 8032 Zürich, Switzerland; dChildren’s ResearchCenter, University Children’s Hospital Zürich, 8032 Zürich, Switzerland; eBioinformatics Core Facility, Swiss Institute of Bioinformatics, 1015 Lausanne,Switzerland; fUnit of Gene Therapy and Stem Cell Biology, Department of Ophtalmology, Jules-Gonin Eye Hospital, University of Lausanne, 1004 Lausanne,Switzerland; gStem Cells and Cancer Group, Vall d’Hebron Institute of Oncology, 08035 Barcelona, Spain; hDepartment of Biochemistry, Faculty of Sciences,University of Geneva, 1211 Geneva, Switzerland; iDepartment of Biological Chemistry and Molecular Pharmacology, Blavatnik Institute, Harvard MedicalSchool, Boston, MA 02115; jComputer-Aided Molecular Engineering, Department of Oncology, Ludwig Institute for Cancer Research, University ofLausanne, CH-1005 Lausanne, Switzerland; and kDepartment of Pathology, Brigham and Women’s Hospital, and Harvard Medical School, Boston, MA 02115

Edited by Iva Greenwald, Columbia University, New York, NY, and approved June 1, 2020 (received for review December 23, 2019)

Notch pathway signaling is implicated in several human cancers.Aberrant activation and mutations of Notch signaling componentsare linked to tumor initiation, maintenance, and resistance tocancer therapy. Several strategies, such as monoclonal antibodiesagainst Notch ligands and receptors, as well as small-moleculeγ-secretase inhibitors (GSIs), have been developed to interferewith Notch receptor activation at proximal points in the pathway.However, the use of drug-like small molecules to target the down-stream mediators of Notch signaling, the Notch transcription acti-vation complex, remains largely unexplored. Here, we report thediscovery of an orally active small-molecule inhibitor (termed CB-103) of the Notch transcription activation complex. We show thatCB-103 inhibits Notch signaling in primary human T cell acute lym-phoblastic leukemia and other Notch-dependent human tumor celllines, and concomitantly induces cell cycle arrest and apoptosis,thereby impairing proliferation, including in GSI-resistant humantumor cell lines with chromosomal translocations and rearrange-ments in Notch genes. CB-103 produces Notch loss-of-functionphenotypes in flies and mice and inhibits the growth of humanbreast cancer and leukemia xenografts, notably without causingthe dose-limiting intestinal toxicity associated with other Notchinhibitors. Thus, we describe a pharmacological strategy that in-terferes with Notch signaling by disrupting the Notch transcriptioncomplex and shows therapeutic potential for treating Notch-driven cancers.

Notch | small-molecule inhbitor | cancer

Transcription factors (TFs) are key mediators of cellularprocesses and cell states. In cancer, TFs are commonly

deregulated indirectly by aberrant upstream signaling cascadesor directly by pathogenic mutations and or translocations. Al-though in principle an attractive class of therapeutic targets, TFsare largely considered undruggable due to the absence of surfacepockets amenable to effective targeting with small molecules.Thus, most currently available targeted cancer therapeutics aimat inhibiting oncogenic signaling pathways at the most proximalpart of the cascade, either through antibodies against ligands orsurface receptors, or using small molecules that inhibit the en-zymatic activities of receptor-associated kinases.The Notch signaling cascade is one example of a pathway that

has emerged as a rational therapeutic target in several cancers.In the adult, Notch signaling in progenitor and stem cells regu-lates tissue homeostasis, self-renewal, and differentiation inseveral organs and cell types, including the intestine, vasculature,and hematopoietic system (1).Notch signaling is initiated through the interaction of a re-

ceptor and ligand on neighboring cells. This event results in

sequential proteolytic cleavages of the receptor mediated bymetalloproteases of the ADAM family and the γ-secretasemultiprotein complex that liberate the Notch intracellular do-main (NICD). NICD subsequently traffics to the nucleus, bindsthe TF RBPJ, and recruits other coactivators, such as master-mind proteins (MAML1-3) and p300, forming a transcriptionactivation complex that initiates the expression of downstreamtarget genes (2).Next-generation sequencing of cancer genomes has identified

numerous oncogenic gain-of-function mutations in NOTCH1 orNOTCH2 in B and T cell leukemias and lymphomas (3–5) andsolid cancers, such as adenoid cystic carcinoma (6) and breastcarcinoma (7, 8). These mutated or truncated genes encodeproteins that are processed to NICD constitutively or have in-creased stability in their active forms, increasing the expression

Significance

The Notch signaling cascade is deregulated by oncogenic le-sions in human cancers and has therefore become an attractivetherapeutic target. Inhibitory monoclonal antibodies and small-molecule γ-secretase inhibitors have been developed to targetthe pathway at the most proximal point of the cascade. Majorhurdles to the therapeutic application of these Notch inhibitorshave been prevalent dose-limiting toxicities in the intestine.Here we report identification and preclinical validation of asmall molecule (CB-103) that inhibits the pathway at the levelof the Notch transcription complex without causing intestinaltoxicity. Its properties and mechanism of action provide CB-103with a more favorable therapeutic index than other types ofNotch targeting agents, a feature that is currently being testedin cancer patients.

Author contributions: R.L., S.C.B., H.G.P., B.B., M.G.-G., Y.A., J.-P.B., and F.R. designedresearch; R.L., J.Z., M.V., C.U., V.F., L.C., A.B., I.C., S.L., S.H.C., U.K., H.G.P., and V.Z. per-formed research; R.L., J.Z., M.V., C.U., V.F., N.Z., L.C., A.B., I.C., S.L., S.H.C., U.K., S.C.B.,H.G.P., B.B., M.G.-G., Y.A., V.Z., J.C.A., J.-P.B., and F.R. analyzed data; and R.L., J.Z., N.Z.,U.K., S.C.B., M.G.-G., Y.A., V.Z., J.C.A., and F.R. wrote the paper.

Competing interest statement: R.L. and F.R. are cofounders of Cellestia Biotech AG, andR.L., M.V., and C.U. are employees of the company.

This article is a PNAS Direct Submission.

Published under the PNAS license.

Data deposition: The SLAM-seq data reported in this paper have been deposited in theGene Expression Omnibus (GEO) database, https://www.ncbi.nlm.nih.gov/geo (accessionno. GSE148228).1To whom correspondence may be addressed. Email: freddy.radtke@epfl.ch.

This article contains supporting information online at https://www.pnas.org/lookup/suppl/doi:10.1073/pnas.1922606117/-/DCSupplemental.

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of target genes that deregulate cell growth and survival. Severalstrategies, such as monoclonal antibodies (MAbs) against Notchligands (9–11) and receptors (12, 13), and small-moleculeγ-secretase inhibitors (GSIs) (14, 15), have been developed toblock Notch receptor activation at proximal points in the path-way. Whereas MAbs have the advantage of specifically inhibitingindividual ligands or receptors, GSIs are pan-Notch inhibitorsthat block signaling through all four Notch receptors (16). GSIswere originally developed for treating Alzheimer disease becausethey inhibit the cleavage of amyloid precursor protein. In addi-tion, they also block the proteolytic cleavage step (S3 cleavage)that generates NICD, leading to their widespread experimentaluse as Notch inhibitors (1, 14). However, GSIs also block theprocessing of more than 90 other substrates, which may com-plicate the interpretation of results produced by GSIs (16). Al-though both MAbs and GSIs have shown beneficial effects inpreclinical Notch-driven tumor models and clinical studies (12,17–21), none of these Notch inhibitors have been clinically ap-proved, largely due to on-target dose-limiting toxicities of theintestinal epithelium (22, 23). Treatment of patients with GSIs isfrequently associated with diarrhea, vomiting, and nausea, whichmay be severe (24, 25). To avoid this toxicity, clinical trials inNotch-driven cancers have relied on intermitting dosing of GSIs(14). However, the question remains as to whether intermittentdosing strategies sustain Notch inhibition long enough to achievetherapeutic efficacy.There have also been attempts to target the pathway down-

stream of the γ-secretase–mediated activation of Notch recep-tors. One is based on the finding that truncated forms ofMAML1 that bind the RBPJ–NICD complex but lack the abilityto recruit other coactivators function in a dominant-negativemanner (26–28). Based on this concept, Bradner and col-leagues (29) synthesized a stapled peptide named SAHM1(stapled α-helical peptide derived from MAML1) designed tomimic dominant-negative forms of MAML1. However, de-veloping drug-like stapled peptides as therapeutics remainschallenging due to manufacturing, stability, and pharmacokineticissues. Another approach utilized screens to identify the smallmolecule Mastermind recruitment-1 (IMR-1), which is alsoproposed to have dominant-negative MAML-like activity (30).Finally, a recent report describes the identification of a smallmolecule that blocks the interaction between RBPJ and SHARP,a protein that forms a corepressor complex with RBPJ (31).However, this approach does not inhibit NOTCH signaling, butrather leads to derepression of NOTCH target genes (31). Al-though all of these Notch TF complex-modulating compoundsshow inhibitory activities in cellular assays, it remains to be de-termined whether these inhibitors possess drug-like properties,as none of these compounds have been tested in clinical trials.Here, we report the discovery and preclinical validation of an

orally active small molecule [6-(4-(tert-butyl)phenoxy)pyridine-3-amine, termed CB-103] that interferes with the function of theNotch transcription complex. CB-103 induces loss-of-functionphenotypes in flies and mice and has antitumor activity in xe-nograft models of Notch-addicted human leukemia and carci-noma without causing gut toxicity. CB-103 possesses excellentdrug-like properties and is currently being evaluated in a phaseI clinical trial in cancer patients.

ResultsIdentification of CB-103 as a Notch Inhibitor. Seeking to discoversmall-molecule inhibitors of Notch signaling, we developed acell-based coculture assay amenable to high-throughput screen-ing of chemical compound libraries. This screen utilized a co-culture system consisting of “signal-receiving” HeLa cellsexpressing Notch1 in combination with a Notch-responsive lu-ciferase reporter, and “signal-sending” HeLa cells expressing theNotch ligand Delta-like 4 (DL4) (Fig. 1 A and B).

A screen of 67,253 compounds from commercially availablelibraries identified 341 compounds with Notch inhibitory activity.Computer-aided self-organizing mapping programs that clustercompounds based on structural similarities allowed reduction ofthe number of hits to 98, of which 33 were validated by a sec-ondary screen in the same coculture assay. These compoundswere then assayed for their ability to block signaling driven by adominant active form of the Notch1 receptor (N1-ICD)(Fig. 1C). Importantly, this approach enables identification ofcompounds that act downstream of the γ-secretase–mediated S3cleavage event. Employing this strategy, we identified the com-pound CB-103 (Fig. 1D). To assess whether CB-103 specificallyinhibits Notch1 or is also active against other Notch receptors,we tested the ability of CB-103 to inhibit Notch2-, Notch3-, andNotch4-mediated signaling using the in vitro coculture assay.CB-103 inhibited Notch signaling mediated by each of the re-ceptors tested in a dose-dependent manner (Fig. 1 E and F). Aswas observed for N1-ICD (Fig. 1G), CB-103 was also able toblock the activity of the dominant active forms of Notch2,Notch3, and Notch4 (N2-ICD, N3-ICD, and N4-ICD, re-spectively) (Fig. 1H). Overall, CB-103 inhibited both ligand-dependent and ligand-independent Notch activation in cell-based assays, with IC50 values ranging from 0.9 to 3.9 μM(Fig. 1 F and H); CB-103 did not inhibit Wnt or Hedgehog sig-naling using reporter assays (SI Appendix, Fig. S1).In light of the above data, we hypothesized that CB-103 acts to

prevent Notch-mediated transcription and thus sought to sub-stantiate this prediction. Experiments using a N1-ICD–GFP fu-sion protein indicated that CB-103 does not prevent nucleartranslocation of N1-ICD (Fig. 1I), excluding impaired traffickingas a mechanism of action. An alternative possibility is that itinterferes with a functional assembly of the transcription com-plex. Consistent with this, expression of increasing amounts ofthe cofactor MAML1 counteracted the inhibitory effect of CB-103 (Fig. 1J), suggesting that CB-103 impairs the recruitment andassembly of components of the Notch transcription complex.

CB-103 Inhibits Growth of Notch-Addicted Human T Cell AcuteLymphoblastic Leukemia Cell Lines through Modulation of theNotch Transcription Complex. To further establish CB-103 as abona fide Notch inhibitor, we tested its ability to directly inhibitthe growth of Notch-dependent cancer cells by initially focusingon T cell acute lymphoblastic leukemia (T-ALL). More than50% of T-ALL patients harbor activating NOTCH1 mutationsresulting in increased Notch signaling (3). Treatment of theNOTCH1-mutated human T-ALL cell line RPMI-8402 and theNOTCH3-driven human TALL-1 cell line with CB-103 or a GSIresulted in down-regulation of the Notch target genes HES1,MYC, and DTX1 (SI Appendix, Fig. S2 A and B), as well as down-regulation of NOTCH1 (SI Appendix, Fig. S2C). Interestingly,protein levels of N1-ICD were unaffected by short-term (6 h)treatment with CB-103 (SI Appendix, Fig. S2C), but were sig-nificantly reduced at later time points (24 and 48 h) (SI Appen-dix, Fig. S2 D and E). In contrast, protein levels of RBPJ wereunaffected (SI Appendix, Fig. S2E). Simultaneous treatment ofRPMI-8402 cells with CB-103 and a proteasome inhibitor didnot restore N1-ICD levels, suggesting that CB-103 does notimpact N1-ICD protein levels by enhancing proteasomal degra-dation (SI Appendix, Fig. S2F). As NOTCH1 exhibits positiveautoregulation in T-ALL cells, these results are consistent with afall in N1-ICD levels due to inhibition of NOTCH1 transcription.In addition, CB-103 induced profound cell growth inhibition inboth RPMI-8402 and T-ALL1 cells (SI Appendix, Fig. S2G and H).In contrast, growth of the Notch-independent HeLa cell line wasunaffected by either CB-103 or GSI treatment (SI Appendix, Fig.S2I). Global gene-expression analysis of CB-103-treated NOTCH1-mutated T-ALL cell lines KOPT-K1 and HBP-ALL further con-firmed down-regulation of N1-ICD–driven growth-promoting

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genes, including MYC, the main oncogenic driver directly regulatedby Notch in T-ALL (32, 33) (SI Appendix, Fig. S3).To gain insight into the molecular mechanism underlying CB-

103 inhibition of Notch-mediated transcription, we generatedRPMI-8402 T-ALL cell lines with reduced sensitivity to CB-103

and then investigated a possible mechanism of insensitivity(mutation) by exome sequencing, motivated by the hypothesisthat drug insensitivity could occur through gene mutations af-fecting the drug’s targets (34). Exome sequencing of CB-103insensitive RPMI-8402 T-ALL cells identified a G193R mutation

A B C D

E F

G H

I J

Fig. 1. Identification of CB-103 as an inhibitor of the Notch transcription activation complex. (A) Schematic of the DL4 Notch1 (N1) coculture assay used high-throughput screening in HeLa cells. Ligand-receptor mediated pathway activation was measured using a Notch-responsive luciferase reporter. (B) Assayvalidation of the DL4-N1 coculture screening with indicated Z′ value. Bar graph shows result of one representative 384-well plate, half treated with 10 μM ofthe GSI DAPT and half with vehicle control. (C) Schematic representation of the N1-ICD–driven luciferase reporter assay used to counter screen-validated hitsof the primary screen. (D) Chemical structure of CB-103. (E) Bar graphs show concentration-dependent assessment of CB-103 and GSI (DAPT) to DL4-Notch1–mediated signaling in the RBPJ-driven reporter and coculture assay. Luciferase activity was measured 24 h after treatment. (F) Dose–responsecurves for mNotch1, mNotch2, mNotch3, and mNotch4 activation following treatment of cocultures with CB-103. (G) Bar graph shows concentration de-pendent assessment of CB-103 and GSI (DAPT) to inhibit N1-ICD–mediated, RBPJ-driven reporter assay. (H) Bar graphs show results of a second independentN1-ICD+ PDX model xenografted into NSG mice. Vehicle control and CB-103 treatment groups were subdivided into high (Upper) and low tumor burdengroups (Lower) based on >20% and <2% of T-ALL cells detected in peripheral blood by flow cytometric analysis at treatment initiation. (I) Nuclear localizationof N1-ICD–GFP fusion protein in HeLa cells in the presence of DMSO or CB-103 (representative of three independent experiments). (Magnification, 100×.) (J)N1-ICD–induced luciferase activity in the presence of CB-103 and increasing amounts of MAML1.

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within the BTD domain of RBPJ. Importantly, engineered ex-pression of a V5-tagged RBPJG193R mutant gene in parental RPMI-8402 cells shifted the IC50 for CB-103 from 2.6 μM to >100 μM,whereas expression of V5-tagged WT RBPJ had minimal effects,indicating that this specific single amino acid change is sufficient toconfer insensitivity to CB-103 treatment (Fig. 2).Next, we performed computational docking studies. CB-103

was docked on the NOTCH1 transcription complex/HES1 pro-moter DNA system to determine a possible binding mode on thenative structure (35). Among the calculated binding modes, oneconfirmed the BTD domain of RBPJ as possible binding site forCB-103 and identified several key RBPJ amino acid residues(Fig. 2A). Expression of engineered forms of RBPJ-bearingmutations in these residues—specifically V5-tagged RBPJF196A,V5-tagged RBPJL245A, or V5-tagged RBPJL248A, but a controlmutant V5-tagged RBPJG194R

—in parental RPMI-8402 cellsconferred resistance to CB-103 (Fig. 2 B and C and SI Appendix,Fig. S4). Thus, combined docking and mutational analysis sup-port the binding of CB-103 to this pocket in the BTD domain. Notably,this pocket is also important for binding of the N1-ICD RAM domain

to RBPJ, providing an explanation for how CB-103 impairs the for-mation and activity of the NOTCH1 transcription complex.In further support of this model, CB-103 also interfered with

recovery of N1-ICD in immunoprecipitates prepared fromRPMI-8402 cells expressing V5-WT-RBPJ, but not from cellsexpressing V5-RBP-JG193R protein (Fig. 3 A and B). Further-more, CB-103 reduced the occupancy of RBPJ and N1-ICD ongenomic Notch-response elements associated with the Notch targetgenes HES1, DTX1, and MYC in RPMI-8402 cells expressingV5-WT-RBPJ but not in cells expressing the V5-RBP-JG193R mutant(Fig. 3C). Taken together, these results strongly suggest that CB-103inhibits Notch-mediated transcription by interfering with assemblyof the Notch transcription complex.The majority of Notch/RBPJ binding sites that mediate acute

changes in gene expression are found in enhancers (36). Thesegenomic response elements are of two distinct types, one con-taining monomeric RBPJ sites and the second dimerichead-to-head RBPJ sites separated by 15 to 17 base pairs, anelement called a sequence-pair site (SPS) that supports loadingof dimeric Notch TF complexes. SPS-mediated Notch signaling

A

B

C

Fig. 2. Single amino acid mutations within the BTD domain of RBPJ cause unresponsiveness to CB-103 in RPMI-8402 cells. (A) Experimental structure of the NOTCH1transcription complex on the HES1 promoter DNA sequence (PDB ID code 3V79) (64). RBPJ (orange), domains are show in ribbon representation. HES1 backbone isshown in ribbon representation, with nucleotides displayed as tubes. Green arrows indicate predicted amino acids important for binding of CB-103 within the BTDdomain of RBPJ. (B) Graph shows dose–response curves of CB-103–treated parental, RPBJwt

–V5- and RBPJG193R–V5-, RBPJL245A–V5-, RBPJL248A–V5-, RBPJF196A–V5-, andRBPJG194R–V5-expressing RPMI-8402 cells. Cells were treated with CB-103 for 3 d. IC50 values for respective cell lines are indicated. (C) Bar graphs show percentage ofapoptotic cells of parental, RBPJwt

–V5- and RBPJG193R–V5-, RBPJL245A–V5-, RBPJL248A–V5-, RBPJF196A–V5-, and RBPJG194R–V5-expressing RPMI-8402 cells treated withDMSO or CB-103 (10 μM) for 72 h. Statistical analysis was performed using two-tailed t test (***P < 0.0005, **P < 0.007; ns, not significant).

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is important for T cell maturation and leukemic transformationbut dispensable for T cell fate specification in mice (37, 38). Todetermine whether CB-103 preferentially inhibited elementscontaining SPSs versus head-to-tail–oriented RBPJ binding sites,we performed luciferase reporter gene assays. Although SPS-driven reporters elicited a much stronger signal compared tohead-to-tail–oriented RBPJ-driven promoters, both were equallysensitive to CB-103. Thus, CB-103 inhibits both monomeric anddimeric Notch1 TF complexes, which agrees with the proposedmode of action of CB-103 (SI Appendix, Fig. S5).Next, we performed kinetic gene-expression analysis using

SLAM-seq [thio(SH)-linked alkylation for the metabolic se-quencing of RNA] on vehicle and CB-103–treated RPMI-8402T-ALL cells to investigate potential differences in the CB-103sensitivity of promoter- and enhancer-driven Notch target genes.Pathway analysis from the Hallmark collection revealed E2Ftargets, MYC targets, and PI3K AKT MTOR signaling as beingthe most rapidly down-regulated pathways. Interestingly, the Notchtarget genes DTX1, HES1, NOTCH3, and NOTCH1, which areregulated by response elements found in promoters or intragenicenhancers, were down-regulated faster than target genes that areregulated by long-distance enhancers—such as MYC, GIMAP1, -5,-6, and -8—suggesting that different Notch target genes may havevarying sensitivities and or kinetics of response to CB-103 (SI Ap-pendix, Fig. S6).In previous studies, the stapled peptide and small molecules

SAHM1 and IMR-1 were also claimed to target the Notch tran-scription complex (29, 30); we therefore compared their activitieswith CB-103 in reporter gene assays and in Notch-driven T-ALLcells. The activity of SAHM1 in reporter gene assays was in-distinguishable from unstapled control peptide; and althoughSAHM1 was cytotoxic, unlike CB-103, it did not down-regulateMYC expression in RPMI-8402 T-ALL cells (SI Appendix, Fig.

S7). In the same assays, we failed to identify any effect of IMR-1 onMYC levels or RPMI-8402 cell growth at doses up to 10 μM (SIAppendix, Fig. S7). Thus, CB-103 acts by a markedly differentmechanism than SAHM1 and IMR-1 with respect to the expectedactivities of direct Notch transcription complex.

CB-103 Function In Vivo Recapitulates Genetic Notch Loss-of-FunctionPhenotypes without Causing Gut Toxicity. Prior to investigating thein vivo activity of CB-103, we profiled the compound to de-termine its drug-like and ADME/PK (absorption, distribution,metabolism, excretion, and pharmacokinetic) properties (SIAppendix, Fig. S8). Then we assessed CB-103’s ability to affect avariety of Notch-dependent cellular processes. First, we studiedthe effect of CB-103 on the development of mechanosensoryorgans in Drosophila, which consist of four distinct lineages(socket, shaft, sheath, and neuron) derived from a single sensoryorgan precursor cell (39). Notch signaling loss induces aberrantsensory organ lineages, including lineages composed of twoneurons and no sheath cells, indicative of sheath-to-neurontransformation (39). Treatment of developing sensory organswith CB-103 resulted in increased numbers of neurons, similar tothe effect produced by GSI (DAPT), demonstrating that CB-103inhibits Notch-dependent cell fate specification in flies (SI Ap-pendix, Fig. S9).We extended these findings to mammals and investigated how

CB-103 affects five Notch-dependent phenotypes in mice: 1)Development of splenic marginal zone B (MZB) cells, 2) thymicT cell development, 3) generation of Esam+ dendritic cells, 4)sprouting of endothelial cells, and 5) induction of goblet celldifferentiation in the small intestine. Each of these processesstrictly require Notch signaling (40–46). As expected, treatmentwith CB-103 (SI Appendix, Fig. S10A) resulted in a dramatic,reversible reduction of B220+CD21hiCD23int MZB cells (SI

RPMI-8402

WTWT RBPJ -V5 RBPJ -V5G193R G193R

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RBPJ - V5 RBPJ - V5MYC

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input inputIP: V5 IP: V5

antibody: N1-ICD N1-ICD 5V5V GgIGgI

RBPJ -V5 WT RBPJ -V5G193R RPMI-8402C0.2

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Fig. 3. CB-103 inhibits assembly of the RBPJ–NICD transcription complex. (A and B) RPMI-8402 cells expressing either RBPJwt-V5 (A) or RBPJG193R-V5 mutantprotein (B) were treated with vehicle control (−) or CB-103 (+), 10 μM for 14 h and subjected to immunoprecipitation using a V5-specific antibody. N1-ICDimmunoprecipitates were assessed by Western blot. Western blot analysis for MYC expression was performed on input: Parental, RBPJwt-V5, or RBPJG193R-V5mutant protein expressing RPMI-8402 cells. (C) DMSO- or CB-103–treated RPMI-8402 cells expressing either RBPJwt-V5 or RBPJG193R-V5 protein were subjectedto ChIP. RBPJ binding regions from NOTCH target genes HES1, DTX1, and MYC were PCR-amplified from input and precipitated DNA. Location of the PCRamplicons is schematically illustrated to the left (red dash). Results are expressed as percentage relative to input. Shown are mean ± SD (n ≥ 3). Statisticalanalysis was performed using one-way ANOVA (****P < 0.0001, **P < 0.009; ns, not significant).

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Appendix, Fig. S10B), mimicking phenotypes caused by loss ofNotch2 (38) or Dll1 (39). Osmotic pump-mediated delivery tomaintain sustained exposure to CB-103 resulted in a concentration-dependent reduction of MZB cells and revealed that a level of700 ng/mL (2.8 μM) in plasma is sufficient to inhibit Notch signaling(SI Appendix, Fig. S10 C and D). Similarly, we observed impairedthymic T cell development (SI Appendix, Fig. S11A), reducednumbers of Esam+CD11c+CD8− dendritic cells (SI Appendix, Fig.S11B), and increased endothelial cell sprouting in CB-103–treatedWT mice (SI Appendix, Fig. S12).In notable contrast, CB-103 treatment did not produce the

expected intestinal phenotype. Genetic deletion of genesencoding Notch1 and Notch2 receptors or Dll1 and Dll4 ligandsor Rbpj, as well as sustained treatment with potent GSIs, result ingoblet cell metaplasia and reduced proliferation of crypt pro-genitor cells (40, 41, 47). Goblet cell metaplasia-associated in-testinal toxicity is one of the main dose-limiting “adverse events”in clinical trials using Notch-targeting agents, such as GSI orantagonistic Notch receptor antibodies (1). We treated mice withvehicle control, CB-103, or the GSI LY3039478. Mice treatedwith vehicle control or CB-103 were analyzed after 1 wk and 4 wkof treatment, while mice treated with LY3039478 were analyzedafter 1 wk due to treatment-related morbidity. Alcian blue andKi67 staining revealed a dramatic increase in goblet cell numbersand a highly significant reduction in crypt cell proliferation inLY3039478-treated animals. In contrast, CB-103–treated ani-mals showed only a moderate increase in goblet cell numbersand a moderate reduction in Ki67

+ crypt cells (Fig. 4).To investigate the basis of the milder gut phenotype in CB-

103–treated mice, we studied its effects on key downstreamtarget genes. In the small intestine, Notch signaling directlyregulates the expression of the stem cell marker gene Olmf4 andmembers of the Hes family of TFs, which repress the transcrip-tional master regulator for secretory cells, Atoh1 (48–50). In situhybridization studies revealed that the expression of Olmf4 andHes1 was nearly completely abrogated by CB-103 and LY3039478,thereby demonstrating Notch inhibitory activity of these agents inthe gut. In contrast, LY3039478-treated animals had significantlyhigher levels of Atoh1 transcripts compared to vehicle- and CB-103–treated animals, which showed only a small increase in Atoh1transcripts relative to vehicle-treated animals (Fig. 4B). To excludepossible confounding influences of variation in absorption orpharmacokinetic properties in vivo, we tested these compounds inintestinal organoid cultures created fromWTmice. LY3039478-treatedorganoids had a very different morphology than CB-103–treated cul-tures and showed increased goblet cell differentiation and decreasedproliferation compared to vehicle- or CB-103–treated organoids (SIAppendix, Fig. S13A).We again observed strong inhibition of the Notchpathway, as indicated by down-regulation of direct Notch target genesOlmf4, Hes1, Hes3, and Hes5 to a similar degree by both CB-103 andLY3039478, whereas Atoh1 transcripts were up-regulated to higherlevels in LY3039478-treated organoids (SI Appendix, Fig. S13B). Theseresults show that while both compounds block Notch signaling in theintestine in vivo and in intestinal organoids, goblet cell metaplasia andprofound growth inhibition were only observed with LY3039478treatment, presumably due to the higher expression levels of Atoh1.

CB-103 Blocks Tumor Growth of GSI-Resistant Cancers. A potentialadvantage of a small-molecule inhibitor of the Notch TF com-plex is the ability to block Notch signaling in tumors that areresistant to Notch-directed MAbs or GSIs by virtue of Notchgene rearrangements that lead to γ-secretase independent Notchactivation. As a proof-of-principle, we studied via lentiviral ex-pression of constitutively nuclear N1-ICD whether CB-103 couldinhibit growth of Notch-dependent human DND-41 T-ALL cellsrendered resistant to agents (such as GSI) that act upstream ofthe Notch TF complex. As expected, parental DND-41 cellsexhibited reduced growth inhibition and Notch signaling following

treatment with either LY3039478 or CB-103, whereas only CB-103elicited growth arrest in DND-41 cells expressing N1-ICD (SI Ap-pendix, Fig. S14A). CB-103 sensitivity was in turn rescued by ectopicexpression of MYC (SI Appendix, Fig. S14B), consistent with priorwork implicatingMYC as a key Notch target gene in T-ALL cells (51).Next, we investigated the ability of CB-103 to inhibit growth of

Notch-dependent cancers that are resistant to MAbs and GSItherapy. We focused on triple negative breast cancer (TNBC),since ∼10% have rearrangements in NOTCH1 and/or NOTCH2that lead to constitutive, ligand-independent Notch activation (7,8). As predicted, growth of the GSI-resistant HCC1187 cell linecarrying a NOTCH2 rearrangement (7) was inhibited by CB-103treatment but not by RO4929097, a GSI previously assessed inclinical phase I/II studies (8) (Fig. 5A). We subsequently estab-lished a stable luciferase reporter line to determine the ability ofCB-103 to inhibit growth of HCC1187 following xenotransplantation.CB-103–treated animals showed remarkable growth inhibition andreduced tumor burden compared to vehicle-treated animals (Fig. 5B).CCDN1, a known Notch target in breast cancer (52, 53), was down-regulated in tumors from CB-103–treated animals compared to con-trols (Fig. 5C). Moreover, CB-103–mediated tumor growth inhibitionwas accompanied by reduced Ki67 and increased Caspase3 staining, while CD31 staining was similar in vehicle- and CB-103–treated animals (SI Appendix, Fig. S15). Our data provideproof-of-concept that CB-103 can inhibit growth of “Notch-addicted” cancer cells expressing mutated forms of Notch thatcannot be targeted with agents that act upstream of the NotchTF complex. Furthermore, patient-derived xenotransplanation(PDX) experiments using a NOTCH1+ oxaliplatin-resistant co-lorectal cancer sample (54) revealed that CB-103 resensitizes thistumor to oxaliplatin treatment in vivo (Fig. 5D).To extend these findings to primary human cancers, we in-

vestigated the ability of CB-103 to inhibit the growth of 19 pri-mary T-ALLs, the human tumor with the highest frequency ofNotch gain-of-function mutations, in a coculture model (47).Dose–response profiles indicated that CB-103 induced growthinhibition in ∼50% of the cases tested with IC50 values in thesubmicromolar range (Fig. 5E). Importantly, the ability of CB-103 to reduce tumor growth correlated strictly with Notch acti-vation status, as only tumors containing N1-ICD responded toCB-103 treatment. Furthermore, growth inhibition induced byCB-103 was associated with the presence of elevated levels ofN1-ICD pretreatment and decreased N1-ICD levels posttreat-ment, but did not strictly correlate with the mutation status ofNOTCH1 or FBXW7 (Fig. 5F). These results indicate that CB-103 selectively inhibits the growth of T-ALLs with ongoingNOTCH1 activation and supports prior studies suggesting thatthe level of N1-ICD predicts tumor response to Notch pathwayinhibitors (44). In line with these ex vivo results, CB-103 pro-longed the survival of mice bearing a NOTCH1-mutated T-ALLPDX model compared to vehicle treatment (Fig. 5 G, Left), anantitumor effect that was also associated with decreased N1-ICDlevels (Fig. 5 G, Right). A second independent N1-ICD+ T-ALLPDX model also showed significantly reduced tumor burden inmice with either high or low tumor burden (>20% and <2%leukemic blasts in the peripheral blood) at treatment initiation,as indicated by percentages of circulating huCD45+CD7+

T-ALL blasts after CB103 treatment (Fig. 5H).

DiscussionIn the last decade unbiased next-generation sequencing efforts ofhuman cancer specimens have identified activating genetic ab-errations in NOTCH genes in a broad spectrum of cancers, in-cluding T-ALL, adenoid cystic carcinoma, chronic lymphocyticleukemia, MZB cell lymphoma, and breast cancer (3, 6, 7, 55).Moreover, many preclinical studies have implicated Notch signalingin almost all hallmarks of cancer, highlighting why this signalingpathway is an intriguing but complex therapeutic target (56).

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Herein we describe a cell-based high-throughput screen thatled to the identification and characterization of a small-moleculeinhibitor (CB-103) of the Notch cascade. CB-103 is a pan Notchinhibitor as it can block Notch-mediated signaling of all fourNotch receptors (Fig. 1), similar to GSIs. Commonly used GSIsblock Notch signaling by inhibiting the proteolytic activity of theγ-secretase multiprotein complex, which cleave Notch receptorsat the S3 site within the transmembrane domain. In contrast toGSIs, CB-103 evidently inhibits the pathway at the most down-stream level—the Notch transcriptional complex—based on itsability to block GSI-insensitive dominant active forms of NICD.Further evidence comes from the generation of a T-ALL cell linethat is resistant to CB-103. The rationale of this experimentaldesign was that drug insensitivity can be afforded through mu-tations that circumvent the inhibitory activity by the drug, mu-tations that have the potential to reveal mechanistic insights bothinto the actions of the drug and its target (34). In the past thisapproach has been used successfully to identify drug targets forBI 2536, a Polo-like kinase1 inhibitor (50), as well as for theproteasome inhibitor Bortezomib, which is clinically used to treatmultiple myeloma and mantle cell lymphoma (34, 57). Usingtranscriptome sequencing analysis, we identified a G193R mu-tation within the RBPJ gene, which encodes an essential com-ponent of the Notch transcription complex; this mutation confersresistance to CB-103 when introduced into Notch-driven T-ALLcells (Fig. 2). Notably, this mutant abrogated the ability of CB-103 to inhibit the formation of Notch transcription complexes, asassessed by immunoprecipitation and chromatin immunopre-cipitation (ChIP) on endogenous Notch-response sites near

target genes, such as HES1, DTX1, and MYC (Fig. 3). In addi-tion, computational docking studies identified the BTD domainof RBPJ as potential binding pocket for CB-103, and accuratelypredicted amino acids residues in RBPJ that when mutatedconfer resistance to CB-103. Taken together, the computationaldocking studies, mutational analysis, as well as pulldown andChIP experiments are in agreement with CB-103 acting as adirect inhibitor of the Notch transcription complex.Oncogenic Notch signaling can be triggered by a variety of

mutations, including single nucleotide substitutions, small in-sertion/deletion mutations, and rearrangements of Notch genes.One advantage of CB-103 over currently available Notch inhib-itors is that it is active against tumor cells bearing any of thesetypes of mutations, which is not uniformly true of other Notchinhibitors. Mutated Notch receptors found in tumors with Notchgene rearrangements lack ectodomains and therefore do not relyon ligand for activation and cannot be targeted with blockingantibodies. Furthermore, ∼5% of TNBCs and 52% of glomustumors have gene rearrangements in NOTCH2 (7, 8, 58) in whichN2-ICD nuclear access is not γ-secretase–dependent, as trans-lational initiation in the aberrant NOTCH2 transcripts lies Cterminal of the S3 cleavage site (7). We performed proof-of-conceptexperiments (Fig. 5 and SI Appendix, Fig. S14) using either engi-neered human T-ALL cell lines expressing N1-ICD or the TNBCcell line HCC1187, which has a NOTCH2 gene rearrangement. No-tably, CB-103 inhibited growth of both cell lines in vitro as well as thegrowth of HCC1187 xenotransplants, whereas GSIs were inactive.Previous studies also identified compounds (SAHM1, IMR-1,

and RIN1) that were claimed to target the Notch transcription

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Fig. 4. CB-103 reduces Notch target gene expression in the small intestine without causing goblet cell metaplasia. (A) Alcian blue (Top row), Ki67 staining (second row), and in situ hybridizations for expression of Notch target genes Olmf4, Hes1, and Atoh1 of representative sections from the proximal smallintestine of vehicle, CB-103– (20 mg/kg 2× day) and GSI- (LY3039478, 20 mg/kg 2× day) treated mice are shown. Mice were treated with CB-103 up to 4 wk andanalyzed at either 1 or 4 wk postadministration. Analysis at two time points revealed comparable results. CB-103 treatment at 4 wk and GSI treatment at 1 wkis depicted. (B) Bar graphs show quantification of indicated stainings and in situ hybridizations. The number of Alcian blue-positive cells per crypt-villus unitand Ki67

+ cells per crypt is expressed as percentage of positive cells per field, of vehicle (n = 2 mice, 130 crypt-villus units for Alcian blue and 462 crypts forKi67), CB-103 (n = 6 mice, 375 crypt-villus units and 430 crypts for Ki67), and GSI (n = 3 mice, 135 crypt villus-units and 330 crypts for Ki67) -treated animals. Insitu hybridization of Olmf4, Hes1, and Atoh1 expression was quantified and is expressed as percentage of positive crypts for Olmf4 (score 4, n = 2 mice forvehicle-treated animals, 190 crypts; n = 6 mice for CB-103 treatment, 600 crypts; n = 3 mice for GSI-treated animals, 300 crypts), percentage of positive cells percrypt for Hes1(score 4, n = 2 mice for vehicle-treated animals ,140 crypts; n = 6 mice for CB-103 treatment, 170 crypts; n = 3 mice for GSI-treated animals, 110crypts) and percentage of positive cells per field for Atoh1 expression (n = 2 mice, vehicle group, 150 crypt-villus units; n = 6 mice, CB-103 group, 340 crypt-villus units; n = 3 mice, LY3039478 group, 220 crypt-villus units). (Scale bars for Alcian blue, 50 μm; for other slides, 20 μm.) Statistical analysis was performedusing unpaired t test (****P < 0.0001; ns, not significant).

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Fig. 5. CB-103 impedes growth of NOTCH+ cancer cell lines and primary human T-ALL. (A) Growth kinetics of HCC-1187 cells treated with DMSO, GSI(RO4929097), and CB-103 (10 μM each) for 6 d. (B) Luciferase-expressing HCC-1187 cells were subcutaneously transplanted into NOD/SCIDγc−/− (NSG) mice andtreated with either vehicle (n = 6) or CB-103 (n = 6) for 15d (2× a day). Bioluminescence was measured 30 d posttransplantation. HCC-1187 tumor volume(mm3) measured over time in xenotransplanted mice treated with either vehicle or CB-103 (25 mg/kg) administrated twice a day (n = 6 for each group) isshown. Two independent experiments were performed. Statistical analysis was performed using two-way ANOVA, ****P < 0.00001. (C) Representative H&Estaining (Left) and immunostaining for Cyclin D1 (Right) of tumors harvested from vehicle- and CB-103–treated animals is shown. (D) Oxaliplatin-resistant M43colorectal cancer cells were transplanted into NSG mice and subsequently treated with either vehicle (n = 18), CB-103 (n = 18), oxaliplatin, or oxaliplatin andCB-103 for 12 d (25 mg/kg 1× a day) and tumor growth fold-change was monitored over time (Left) and at end stage of the experiment (Right). One-wayANOVA, Tukey’s multiple comparisons test *P < 0.05; ***P < 0.0003; ****P < 0.0001; ns, not significant. (E) Response to CB-103 of T-ALL cells in vitro. Patient-derived xenografts (n = 20) were maintained on human mesenchymal stromal cells and incubated with 0.01, 0.1, 1, 10, or 25 μM CB-103 for 7 d. Graphrepresents IC50 values of each patient sample treated with CB-103. Blue circles represent patient samples positive for the presence N1-ICD, red circles representpatient samples negative for N1-ICD. (F) Immunoblots underneath show N1-ICD levels decrease upon treatment with CB-103. N1-ICD detection in indicatedpatient samples (numbered) on Western blot after 72-h exposure to DMSO or CB-103 is shown. Mutation status for NOTCH1 and FBXW7 are indicated. (G)Event-free survival analysis after treatment of leukemia xenografts of the T-ALL corresponding to case 1. Kaplan–Meier survival curve is shown for xeno-grafted NSG mice. Treatment with vehicle or CB-103 as indicated when 10% of T-ALL cells were detected in peripheral blood by flow cytometry. Log-rank(Mantel–Cox) test and P value as indicated. Bar graph represents detection levels of N1-ICD in T-ALL cells after in vivo treatment with CB-103. The mean ratio(±SEM) of N1-ICD (Val-1744) determined in vehicle and CB-103–treated animals (two per condition). A representative Western blot example for N1-ICD oftreated animals is shown. (H) Bar graphs show results of a second independent N1-ICD+ PDX model xenografted into NSG mice. Vehicle control and CB-103treatment groups were subdivided into high and low tumor burden groups based on >20% and <2% of T-ALL cells detected in peripheral blood by flowcytometric analysis at treatment initiation. Animal groups were treated for 12 d with either vehicle control or CB-103. Bar graphs show absolute numbers ofhuCD45+CD7+ T-ALL cells after treatment. Animals with high tumor burden: n = 5 for vehicle control and n = 3 for CB-103–treated animals; animals with lowtumor burden: n = 5 for both vehicle and CB-103 treated animals. Statistical analysis: Student’s t test test, *P < 0.05.

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complex (29–31). However, in reassessing the activity ofSAHM1, we were unable to confirm any “on-Notch” inhibitoryeffects in reporter gene assays or T-ALL cells (SI Appendix, Fig.S7). IMR-1 is a small chemical compound that was identifiedbased on the strategy of inhibiting MAML1 recruitment to theNotch transcription complex (30). A side-by-side comparison ofCB-103, the GSI LY3039478, SAHM1, and IMR-1 for theirability to block growth of a Notch-driven human T-ALL cell lineshowed that IMR-1 had no effect on Notch target gene expres-sion or cell growth at doses up to 10 μM, whereas CB-103 andGSI were active (SI Appendix, Fig. S7). IMR-1 evidently caninhibit Notch signaling at higher concentrations, as previouslyreported (30, 59, 60). RIN1 is another small molecule that wasrecently reported to modulate the Notch transcription complex(31). This small chemical compound was identified in a reporter-based cell culture assay aimed at identifying inhibitors of in-teractions between RBPJ and SHARP, which is a componentof a transcriptional repressor complex that binds RBPJ in theabsence of NICD. In line with this possibility, RIN1-treatedcells up-regulated Notch target genes, mimicking the effects ofRBPJ knockdown. The activity of RIN1 has yet to be assessed,in regard to therapeutic efficacy and intestinal toxicity (31),and comparative evaluation with CB-103 warrants futureconsideration.One of the major dose-limiting toxicities and hurdles to the

therapeutic application of the pan-Notch inhibitor has been in-testinal toxicity (22, 23). Genetic studies in mice show that Notchacts as a stem and progenitor cell gate keeper and is importantfor secretory versus absorptive cell fate differentiation. Condi-tional, gut-specific inactivation of Notch1 and -2, the ligands Dll1and Dll4, and Rbpj result in the loss of proliferative crypt pro-genitors and conversion into postmitotic goblet cells (40, 41, 47).A similar phenotype has been observed in mice with simulta-neous gut-specific inactivation of the Notch target genes Hes1,Hes3, and Hes5, indicating Notch signaling regulates intestinalhomeostasis at least in part through Hes genes (49). Conversely,transgenic Notch gain-of-function experiments cause a reciprocalphenotype consisting of a block in secretory cell differentiationand an expansion of immature crypt progenitors (61). The gutphenotype created by knockout of Notch pathway components isalso observed in rodents treated with potent GSIs, such asdibenzazepine (47), suggesting that the gut toxicity caused byGSI is mostly driven by Notch inhibition (16). In this context, it isinteresting to note that four different γ-secretase complexes existand that most available GSIs block all complexes, which is likelyto account for the intestinal toxicities in GSI-treated animals orpatients. A recent report showed that selective pharmacologicalinhibition of one (presenilin-1) of the four (PSEN) γ-secretasesubclasses is effective in reducing the leukemic burden of PSEN-1 expressing T-ALL cells in xenotransplantation assays withoutcausing intestinal toxicity (62). Thus, selective inhibition of theγ-secretase complex might be a potential therapeutic strategy forsafely targeting Notch-driven tumors, provided Notch cleavage ismostly mediated by specific PSEN subclasses.These effects of GSI involve alterations in the TF Atoh1 (also

known as Math1), which is a master regulator of the secretorylineage (50, 63). Conditional gut-specific inactivation of Atoh1results in loss of all secretory cells and in the context of GSI-mediated Notch inhibition has been shown to be essential forgoblet cell fate conversion (63).Unexpectedly, in vivo administration of CB-103 to mice did

not lead to the anticipated goblet cell metaplasia phenotype,which was observed in GSI-treated animals. Direct Notch target

genes, such as Olmf4 and Hes1, were both down-regulated byCB-103 and GSI, indicating that CB-103 reached the target tis-sue. In contrast, Atoh1 transcripts were significantly higher inGSI-treated animals (Fig. 4). This distinction was confirmed inintestinal organoid cultures, excluding the possibility that thisresult is a consequence of variation in absorption or pharmacoki-netic properties in vivo. LY3039478-treated organoids displayed avery different morphology than CB-103–treated cultures, showingincreased goblet cell differentiation and decreased proliferationcompared to vehicle- or CB-103–treated organoids (SI Appendix,Fig. S13). The mechanisms underlying this distinction are currentlyunknown. Several possibilities can be considered. First, CB-103inhibits protein–protein interaction and might therefore producemore incomplete Notch inhibition than a potent GSI. A secondpossibility, which is not mutually exclusive, is that CB-103 may in-hibit only a subset of RBPJ complexes, leading to variation in theresponsiveness of different Notch target genes. Nevertheless, inNotch-driven tumors, where the levels of signaling are well abovethat of normal tissues due to activating mutations, CB-103 is evi-dently potent enough to produce responses in preclinical models.Further work is necessary to parse out these distinctions.In summary, our discovery and characterization of CB-103

makes a compelling case that small-molecule inhibitors can bedeveloped and used to block TF complexes, which are down-stream of many aberrant signaling cascades, but have been his-torically intransigent to therapeutic targeting. CB-103 interfereswith the Notch TF complex, and may thereby convey a morefavorable therapeutic window than previous Notch-targetingagents. Motivated by this knowledge and by its pharmacologi-cal characteristics, CB-103 is currently being evaluated in phase I/IIclinical trials (https://clinicaltrials.gov/ct2/show/NCT03422679).

Materials and MethodsDetails of materials and methods are provided in SI Appendix, includingsources of constructs, compounds, cell lines and cell culture conditions (exvivo, in vitro), luciferase reporter assay, assays of cell cycle, proliferation, cellviability, stable transformants, and animal studies. Details of microscopy,image processing and analysis, FACS and computational docking studies aredescribed in SI Appendix. Methods for Western blot, immunoprecipitationand ChIP, qRT-PCR, in situ hybridization, histology and immunostaining, RNAsequencing and SLAM-seq, and bioinformatic and statistical analyses are alsoprovided in SI Appendix.

Ethics Statement. All animal work was carried out in accordance with Swissnational guidelines. This study was reviewed and approved by the cantonalveterinary service, Vaud.

Data Availability Statement. All data generated in this study are included inthe paper and SI Appendix. The SLAM-seq data reported in this paper havebeen deposited in the Gene Expression Omnibus (GEO) database, https://www.ncbi.nlm.nih.gov/geo (accession no. GSE148228).

ACKNOWLEDGMENTS. We thank the team of the Biomolecular ScreeningFacility, Ecole Polytechnique Fédérale de Lausanne, Gerardo Turcatti, MarcChambon, Nathalie Ballanfat and Manuel Bueno; Viktoria Reinmüller andJieping Zhu for compound synthesis; Daniel Hall and Rhett Kovall for tech-nical support and discussion of the mode of action of CB-103; and DouglasHanahan and Maximilien Murone for critical reading, discussions, and edit-ing of the manuscript. Molecular graphics and analyses were performed withUniversity of California, San Francisco Chimera (NIH P41-GM103311). Thiswork was in part supported by the Swiss National Science Foundation andthe Swiss Cancer League, the National Centre of Competence in ResearchChemical Biology (F.R.), the foundation “Kind und Krebs,” the “Krebsliga Zur-ich,” the Swiss National Science Foundation (310030-133108), and the clinicalresearch focus program “Human Hemato-Lymphatic Diseases“ of the Universityof Zürich. J.C.A. is supported by Harvard University Ludwig Institute.

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